Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: more ..

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) alkaline phosphatases. IAP expression is largely restricted to the gut, especially to the epithelial cells (enterocytes) of the small intestinal mucosa. The exact biological function of IAP is unknown.

The goal of this HTS is to identify novel and specific activators of IAP. The only known to date class of alkaline phosphatases activators are hydroxyl-containing compounds, such as diethanolamine (DEA), that act as phosphoacceptor substrate and exhibit its effect in high-mM concentration range. Compounds with a similar mode of action are expected to demonstrate diminished stimulating potential if tested in the presence of high concentration of DEA. Therefore, for detection of compounds with diverse mode of action, the HTS campaign was performed in the presence close-to-Km DEA concentration.

IAP screening was developed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). This assay represents a selectivity screening for activators of tissue nonspecific alkaline phosphatase (TNAP) screened at BCCG (AID 1001). RO3 submission, MH082385-01: Activators of the Pyrophosphatase Activity of Alkaline Phosphatase. Assay Providers: Drs. Jose Luis Millan and Eduard Sergienko, Sanford-Burnham Medical Research Institute, San Diego, CA.

Alkaline phosphatase (EC 3.1.3.1) (APs) catalyze the hydrolysis of phosphomonoesters, releasing inorganic phosphate and alcohol. APs are dimeric enzymes found in most organisms. In human, four isozymes of APs have been identified. One isozyme is tissue-nonspecific (designated TNAP) and three other isozymes are tissue-specific and named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) alkaline phosphatases. IAP expression is largely restricted to the gut, especially to the epithelial cells (enterocytes) of the small intestinal mucosa. The exact biological function of IAP is unknown.

IAP is inhibited by a number of inhibitors. They include L-phenylalanine, L-tryptophan, L-leucine and phenylalanine-glycylglycine. While the biological implications of this inhibition are not known, these inhibitors have proven to be useful in the differential determination of AP isozymes as important diagnostic markers in many diseases. However, these known inhibitors of IAP are not entirely specific for IAP isozyme and have milllimolar affinity. In addition, they are common aminoacids that are ubiquitously present in the tissues and involved in diverse metabolic pathways, and therefore, are not appropriate tools for biological studies. Thus, the aim of this MLSCN probe project is to obtain novel chemical scaffolds that can be used as chemical probes for IAP. Bovine enzyme was utilized in this primary screening as a highly active alternative with high homology to the human enzyme.

IAP screening was designed and performed at the Sanford-Burnham Center for Chemical Genomics (SBCCG) as part of the Molecular Library Screening Center Network (MLSCN). The assay was developed as a secondary assay for TNAP probe generation project (AID 518): XO1 submission, MH077602-01, Pharmacological inhibitors of tissue-nonspecific alkaline phosphatase (TNAP), Assay Provider Dr. Jose Luis Millan, Sanford-Burnham Medical Research Institute, San Diego, CA.

IAP activation was calculated using the following formula: Activation Factor (AF) = (Signal_Well - Mean_NC)/(Mean_PC - Mean_NC), where Signal_Well corresponds to luminescence signal in the well with a compound, Mean_NC and Mean_PC correspond to mean values of corresponding controls in the plate. Compounds with greater than or equal to 2-fold activation (AF >= 2) of IAP at 20-uM concentration are defined as actives of the primary screening. To simplify the distinction between the inactives of the primary screen and of the confirmatory screening stage, the Tiered Activity Scoring System was developed and implemented. Its utilization for the IAP assay is described below. Activity ScoringActivity scoring rules were devised to take into consideration compound efficacy, its potential interference with the assay and the screening stage that the data was obtained. Details of the Scoring System will be published elsewhere. Briefly, the outline of the scoring system utilized for the IAP assay is as follows: 1) First tier (0-40 range) is reserved for primary screening data and the score is correlated with IAP activation factor demonstrated by a compound at 20 uM concentration:a. If AF<1, then the assigned score is 0b. For all other AF values, Score = 40 - 40/AFThis formula results in a score that is equal 20 for AF=2 and asymptotically approaches 40 with increasing AF values.2) Second tier (41-80 range) is reserved for dose-response confirmation data 3) Third tier (81-100 range) is reserved for resynthesized true positives and their analogues